Project Summary A central goal in disease biology is to describe the molecular processes responsible for transformation of a cell from a normal state into a pathological one. Compared to the rapid progresses in DNA and RNA sequencing technologies, characterization of the final and arguably most actionable element of the central dogma, protein, has lagged behind. The dynamic relationship between the genome of a cell and its proteome is poorly understood, reflecting multiple layers of transcriptional/post-transcriptional regulation. In particular, the complexity of the human proteome is greatly expanded by the ~400 different types of protein posttranslational modifications (PTMs). The various PTM events, either alone or in combination (i.e., ?cross-talk?), represent powerful mechanisms to modulate the function of a protein (e.g., activity, stability and localization), the collection of which convey information within the signaling network that underlies the complex traits in various pathophysiological conditions. However, because of many inherent technical difficulties associated with the analysis of protein PTMs (e.g., chemically diverse, unstable and low abundance), a complete description of the posttranslationally modified proteome of any given cells remains a daunting task. The overarching mission of our program is to: (1) develop cutting-edge quantitative proteomic approaches to systematically identify and characterize novel PTMs, (2) comprehensively interrogate the signaling events regulated by phosphorylation (mTOR pathways) and ADP-ribosylation (PARP pathways), and (3) combine these systems biology approaches with classical biochemical, cell biology and animal experiments to decipher the molecular underpinnings of cell growth and stress responses that are controlled by these two important pathways. To accomplish these goals, we will leverage our preliminary results (including a large set of unique hits, reagents and methods), and center our efforts on the following six goals. First, we will develop innovative mass spectrometric technologies with dramatically improved performance for global, quantitative and site-specific analysis of novel PTMs. Second, we will investigate the role of IGFBP5 (a recently identified extracellular target of mTORC1) as a mediator of the ?non-cell autonomous? function of mTORC1. Third, we will determine the role of EGR1 (a novel hit identified from our previous MS screen) as a master regulator of the mTORC1-dependent feedback loops. Fourth, we will generate a tissue-specific atlas of mTORC1 phosphorylation substrates, and in doing so, interrogate non-uniform effects of this important pathway on the physiology of different tissues. Fifth, we will develop a large-scale MS approach to site-specific characterization of the D/E-mono-ADP-ribosylated proteome, and finally we will develop a large-scale method to measure absolute protein PARylation stoichiometries. Together, these studies provide a comprehensive framework for the MS identification and functional characterization of PTMs events linked to cell growth control and stress responses.